Methods of processing collagen from animal tissue

Abstract

Processes are provided that yield high molecular weight collagen without enzymatic or chemical treatment, preserving triple helical content of native collagen. Processing conditions and energy input on animal tissue supply streams can be controlled to obtain collagen compositions with desirable yields and properties, including ratios of denatured and nondenatured collagen with reduced processing time and higher mass throughput.

We claim: 
     
         1 . A method for extracting collagen from isolated animal tissue, said method comprising:
 non-thermal mechanically and/or hydrodynamically shearing an animal tissue that comprises collagen in an aqueous media, thereby separating a collagen-containing liquid fraction comprising extracted collagen from a solid fraction, wherein the shearing is conducted under controlled conditions of temperature, pressure, and hydrodynamic energy such that at least about 5% of the extracted collagen in the collagen-containing liquid fraction is in a nondenatured form.   
     
     
         2 . The method of  claim 1 , wherein greater than about 50% of the extracted collagen in the collagen-containing liquid fraction is in the nondenatured form. 
     
     
         3 . The method of  claim 1 , wherein the extracted nondenatured collagen comprises a molecular weight greater than about 220 kDa. 
     
     
         4 . The method of  claim 1 , wherein at least about 5% of the extracted collagen in the collagen-containing liquid is in a denatured or hydrolyzed form. 
     
     
         5 . The method of  claim 1 , wherein greater than about 50% of the collagen in the collagen-containing liquid is in the denatured or hydrolyzed form. 
     
     
         6 . The method of  claim 1 , wherein the collagen-containing liquid fraction comprises greater than about 5% of the collagen content in the isolated animal tissue from which it is extracted. 
     
     
         7 . The method of  claim 1 , wherein said isolated animal tissue is maintained at a temperature that is less than about 10° C. prior to mechanical and/or hydrodynamic shearing. 
     
     
         8 . The method of  claim 7 , wherein microbial or enzymatic degradation of collagen or activation of endotoxins is limited in comparison to an isolated animal tissue raw material that is not maintained at a temperature less than about 10° C. 
     
     
         9 . The method of  claim 1 , wherein said mechanical and/or hydrodynamic shearing of the isolated animal tissue that comprises collagen comprises one or more of:
 (i) low shear extrusion, optionally at about 100 sec −1  or less, through an extruder that comprises an inlet, a compression screw, a barrel, and a positive displacement pump, wherein the tissue is pumped through the barrel with the compression screw under pressure of about 10 psi to about 150 psi and with temperature conditions controlled such that the difference in temperature between the inlet and outlet does not vary more than about 20° C., wherein extruded material that flows through the outlet is further separated into the collagen-containing liquid fraction that comprises extracted collagen and the solid fraction;   (ii) compression in a perforated compression cylinder, wherein the cylinder is compressed at a pressure of about 10 psi to about 150 psi and a cycle time of about 5 sec to about 30 sec, wherein the collagen-containing liquid fraction flows through perforations on the periphery of the cylinder; or   (iii) processing through a dewatering press apparatus at a pressure of about 10 psi to about 150 psi and a residence de-watering time of about 5 sec to about 30 sec.   
     
     
         10 . The method of  claim 9 , wherein the collagen-containing liquid fraction and/or the solid fraction is further processed under the same or different conditions in the same or different apparatus to improve yield and/or purity of the extracted collagen. 
     
     
         11 . The method of  claim 1 , wherein prior to shearing the isolated animal tissue, said tissue is subjected to an input of electric field and/or acoustic energy that does not substantially degrade the structure of the collagen extracted from the tissue. 
     
     
         12 . The method of  claim 11 , wherein the input energy is provided by pulsed electric field and/or ultrasonication treatment. 
     
     
         13 . The method of  claim 11 , wherein the input of energy comprises pulsed electric field treatment at an electric field strength that is about 3 kV/cm to about 90 kV/cm. 
     
     
         14 . The method of  claim 11 , wherein the input of energy comprises ultrasonic treatment at about 20 Hz to about 40 Hz and about 150 W to about 250 W instrument input power. 
     
     
         15 . The method of  claim 11 , wherein the input of energy comprises about 40 kJ/kg to about 100 kJ/kg of the isolated animal tissue. 
     
     
         16 . The method of  claim 11 , wherein the input of energy does not raise the temperature of the isolated animal tissue or a processing milieu in which it resides above about 30° C. 
     
     
         17 . The method of  claim 1 , wherein prior to mechanical and/or hydrodynamic shearing the tissue, the isolated animal tissue is mechanically diced, macerated, minced or ground, thereby processing the tissue into smaller pieces under low shear conditions. 
     
     
         18 . The method of  claim 17 , wherein the isolated animal tissue is mechanically diced or minced into pieces that comprise an average cross-sectional area of about 0.5 square inches to about 2 square inches. 
     
     
         19 . The method of  claim 11 , wherein prior to the input of energy, the isolated animal tissue is mechanically diced or ground into smaller pieces under low shear conditions. 
     
     
         20 . The method of  claim 19 , wherein the isolated animal tissue is mechanically diced or minced into pieces that comprise an average cross-sectional are of about 0.5 square inches to about 2 square inches. 
     
     
         21 . The method of  claim 1 , wherein said isolated animal tissue comprises skin tissue. 
     
     
         22 . The method of  claim 21 , wherein the skin tissue comprises fish skin. 
     
     
         23 . The method of  claim 1 , wherein the collagen-containing liquid fraction is produced from the isolated animal tissue in less than about 6 hours. 
     
     
         24 . The method of any of  claims 1 to 23 , wherein the method does not comprise energy, chemical, and/or enzymatic conditions that intentionally denature or hydrolyze collagen. 
     
     
         25 . The method of  claim 24 , wherein the method does not include conditions that intentionally degrade or hydrolyze collagen to produce fragments that are smaller than about 220 kDa. 
     
     
         26 . The method of any of  claims 1 to 23 , wherein at least about 90% of the method is conducted at a temperature that is lower than the denaturation temperature for the collagen. 
     
     
         27 . The method of  claim 26 , wherein at least about 95% of the method is conducted at a temperature that does not exceed 30° C. 
     
     
         28 . The method of any of  claims 1 to 23 , wherein at least about 70% of the method is conducted at a mechanical shear rate that is below 100 sec −1 . 
     
     
         29 . The method of any of  claims 1 to 23 , wherein the collagen-containing liquid fraction comprises an aerobic plate count for microbial contamination less than about 104 CFU/g. 
     
     
         30 . The method of any of  claims 1 to 23 , wherein the collagen-containing liquid fraction comprises at least about 10 2 -fold reduction in aerobic plate count for microbial contamination in comparison to the isolated animal tissue from which it was extracted. 
     
     
         31 . A collagen-containing liquid prepared by the method of any of  claims 1 to 23 , wherein at least about 5% of the extracted collagen is in the nondenatured form. 
     
     
         32 . The collagen-containing liquid according to  claim 31 , wherein greater than about 50% of the extracted collagen is in the nondenatured form. 
     
     
         33 . A composition comprising isolated collagen, wherein said isolated collagen is extracted collagen that is purified from the collagen-containing liquid according to  claim 31 . 
     
     
         34 . The composition of  claim 33 , wherein the extracted collagen comprises a molecular weight greater than about 220 kDa. 
     
     
         35 . A composition comprising isolated collagen, wherein greater than about 50% of said collagen is in a nondenatured form. 
     
     
         36 . The composition of  claim 35 , wherein the nondenatured collagen comprises a molecular weight greater than about 220 kDa. 
     
     
         37 . A food, nutritional supplement, nutraceutical, animal feed, pharmaceutical dosing, drug delivery or gene carrier formulation, wound or burn care dressing, cosmetic additive in a gel, cream, salve, drop, ointment or topical dressing, cosmeceutical, collagen cell culture scaffold, cultivated meat or meat analogue, additive manufacturing 3-D printable matrix, 3-D bio-ink for tissue engineering, cell carrier, and/or medical device component, or container or packaging material, comprising a composition according to  claim 33 or 35 . 
     
     
         38 . A food, nutritional supplement, nutraceutical, animal feed, pharmaceutical dosing, drug delivery or gene carrier formulation, wound or burn care dressing, cosmetic additive in a gel, cream, salve, drop, ointment or topical dressing, cosmeceutical, collagen cell culture scaffold, cultivated meat or meat analogue, additive manufacturing 3-D printable matrix, 3-D bio-ink for tissue engineering, cell carrier, and/or medical device component, or container or packaging material, comprising a collagen-containing liquid according to  claim 32 . 
     
     
         39 . A method for extracting collagen from isolated animal tissue, said method comprising:
 mechanically and/or hydrodynamically shearing an animal tissue that comprises collagen in an aqueous media, thereby separating a collagen-containing liquid fraction comprising extracted collagen from a solid fraction, wherein the shearing is conducted under controlled conditions of temperature, pressure, and hydrodynamic energy such that at least about 5% of the extracted collagen in the collagen-containing liquid fraction is in a denatured form.   
     
     
         40 . The method of  claim 39 , wherein greater than about 50% of the extracted collagen in the collagen-containing liquid fraction is in the denatured form. 
     
     
         41 . The method of  claim 39 , wherein said mechanical and/or hydrodynamic shearing of the isolated animal tissue that comprises collagen comprises one or more of:
 (i) low shear extrusion, optionally at about 100 sec −1  or less, through an extruder that comprises an inlet, a compression screw, a barrel, and a positive displacement pump, wherein the tissue is pumped through the barrel with the compression screw under pressure of about 10 psi to about 150 psi and temperature about 40° C. to about 60° C., wherein extruded material that flows through the outlet is further separated into the collagen-containing liquid fraction that comprises extracted collagen and the solid fraction;   (ii) compression in a perforated compression cylinder, wherein the cylinder is compressed at a pressure of about 10 psi to about 150 psi, temperature about 40° C. to about 60° C., and a cycle time of about 5 sec to about 30 sec, wherein the collagen-containing liquid fraction flows through perforations on the periphery of the cylinder; or   (iii) processing through a dewatering press apparatus at a pressure of about 10 psi to about 150 psi, temperature about 40° C. to about 60° C., and a residence de-watering time of about 5 sec to about 30 sec.   
     
     
         42 . The method of  claim 39 , wherein prior to shearing the isolated animal tissue, said tissue is subjected to an input of electric field and/or acoustic energy. 
     
     
         43 . The method of  claim 42 , wherein the input energy is provided by pulsed electric field and/or ultrasonication treatment. 
     
     
         44 . The method of  claim 42 , wherein the input of energy comprises pulsed electric field treatment at an electric field strength that is about 3 kV/cm to about 90 kV/cm. 
     
     
         45 . The method of  claim 42 , wherein the input of energy comprises ultrasonic treatment at about 20 Hz to about 40 Hz and about 150 W to about 250 W instrument input power. 
     
     
         46 . The method of  claim 42 , wherein the input of energy comprises about 40 kJ/kg to about 100 kJ/kg of the isolated animal tissue. 
     
     
         47 . The method of  claim 39 , wherein prior to mechanical and/or hydrodynamic shearing the tissue, the isolated animal tissue is mechanically diced, macerated, minced or ground, thereby processing the tissue into smaller pieces under low shear conditions. 
     
     
         48 . The method of  claim 47 , wherein the isolated animal tissue is mechanically diced or minced into pieces that comprise an average cross-sectional area of about 0.5 square inches to about 2 square inches. 
     
     
         49 . The method of  claim 42 , wherein prior to the input of energy, the isolated animal tissue is mechanically diced or ground into smaller pieces under low shear conditions. 
     
     
         50 . The method of  claim 49 , wherein the isolated animal tissue is mechanically diced or minced into pieces that comprise an average cross-sectional are of about 0.5 square inches to about 2 square inches. 
     
     
         51 . The method of  claim 39 , wherein said isolated animal tissue comprises skin tissue. 
     
     
         52 . The method of  claim 51 , wherein the skin tissue comprises fish skin. 
     
     
         53 . The method of  claim 39 , wherein the collagen-containing liquid fraction is produced from the isolated animal tissue in less than about 6 hours. 
     
     
         54 . A collagen-containing liquid prepared by the method of any of  claims 39 to 53 , wherein at least about 5% of the extracted collagen is in the denatured form. 
     
     
         55 . The collagen-containing liquid according to  claim 53 , wherein greater than about 50% of the extracted collagen is in the denatured form. 
     
     
         56 . A food, nutritional supplement, nutraceutical, animal feed, pharmaceutical dosing, drug delivery or gene carrier formulation, wound or burn care dressing, cosmetic additive in a gel, cream, salve, drop, ointment or topical dressing, cosmeceutical, collagen cell culture scaffold, cultivated meat or meat analogue, additive manufacturing 3-D printable matrix, 3-D bio-ink for tissue engineering, cell carrier, and/or medical device component, or container or packaging material, comprising a collagen-containing liquid according to  claim 54 .